Separation, detection and quantification of biomolecules in lab-on-a-chip systems

Since the introduction of the of Micro Total Analysis Systems concept in 1990, important progress in microfluidic analytical systems has been realized. In many cases, microfluidic systems have shown improved analytical performances over classical methods, while consuming small amounts of sample and reagents, and strongly reducing analysis time. In this thesis, we propose to explore two important topics in analytics in the microchip format: (i) Capillary electrophoresis (CE) on-chip, where we will focus on pressure-based sample injection techniques and (ii) the isolation and quantification of biological molecules, like antibodies, using magnetic nanoparticles as support in a sandwich immunoassay. After a general introduction, we will introduce the theoretical concepts for understanding our work in CE on-chip, followed by the description of the magnetism applied to the manipulation of magnetic nanoparticles in a microchannel. The state-of-the-art of the injection techniques in a CE microchip and the utilization of microfluidic chips for immunoassays will be introduced, followed by the presentation of two intensively used microfabrication techniques: (i) the powder blasting micro-erosion process for the realization of CE microchips, and (ii) deep reactive-ion etching for the microfabrication of microfluidic chips used for the retention of magnetic nanoparticles. As main results of this thesis, we develop a novel and promising concept of pressure-based injection in a CE microchip, where the sample is flowing continuously through the microchip using a single potential that is constant in time, and injected by a pressure pulse in less than one second. Then, we will introduce a new and original concept to retain superparamagnetic nanoparticles in a microfluidic chip, using a microchannel with periodically enlarged sections. We use this retention technique for the detection and quantification of monoclonal antibodies spiked in cell culture medium or directly obtained from the production of a cell culture. Thereby, we perform a complete on-chip sandwich immunoassay, while using only nanoliters of sample and reagents. Finally, we will conclude the thesis by giving an outlook of the expected future developments of our work and propose potential new applications in other area of microfluidic analytical microsystems.

High-throughput surface patterning with wafer-sized stencils fabricated by a DUV/MEMS process

Jürgen Brugger, Marc Antonius Friedrich van den Boogaart

The endeavour to develop nanodevices demands for patterning methods in the nanometer scale. The continuous improvement in lithography methods based on deep UV (DUV), X-ray, or electron beam exposure allow for further progress in integrated circuit hardware manufacturing for the coming years. To bring nanodevices to the market, however, there is a need for fast, low-cost replication nanopatterning methods. In addition, an increased flexibility of the nanopatterning methods is important for the engineering of multimaterial and multifunctional nano/micro-electro-mechanical systems (NEMS/MEMS), such as polymer-based electronic and sensor devices, 3D microfluidic systems, and bio-analytical systems. The above-mentioned high-end patterning methods are all based on photolithography and etching, limiting the flexibility in substrate and material combinations. In addition, they have the drawback of the use of high-cost equipment. A series of alternative, complementary surface patterning methods are currently being developed that do not rely on photoresist exposure and thin-film etching steps, e.g. local printing of molecular layers (soft-lithography) and shadow-mask deposition (nanostencil patterning). The nanostencil method is a resistless patterning method based on direct, local deposition of material on an arbitrary surface through a solid-state silicon nitride (SiN) membrane , . We present here the advances towards a full-wafer (100 mm) DUV/MEMS-based nanostencil to allow high-throughput, large-area nanopatterning of mesoscopic structures (10^9 - 10^5 m). The mesoscopic patterns were first defined by a 4× reduced projection exposure using an ASML wafer stepper and then transferred into a SiN layer by means of reactive ion etching (RIE). The membranes were released by wafer-through etching using potassium hydroxide (KOH). Figure 1 shows the mid-process details of 200 nm DUV patterns after the RIE transfer into the 500-nm thick SiN layer. Patterning of nano-scale structures using stencils allows for a large choice of materials and surfaces to be nanopatterned, since it is a non-contact method and no etch steps need to be applied. We have studied the nanopatterning of metals (Al, Au, Bi, and Cr) on various surfaces (silicon, oxides, freestanding SiN cantilevers, and self-assembled monolayers (SAM)) for different applications (molecular electronics, nano-mechanical devices with a 90 MHz resonance frequency , as well as nano-scale Hall-sensor devices). Unconventional substrates for (Bio) MEMS and NEMS have also been applied such as highly ordered pyrolytic graphite (HOPG), polydimethylsiloxane (PDMS), and SU-8.

2004

Magnetic beads retention device for sandwich immunoassay: comparison of off-chip and on-chip antibody incubation

Martinus Gijs, Frédéric Lacharme, Caroline Vandevyver

We use magnetic microbeads, which are magnetically self-assembled in chains in a microfluidic chip, as reaction substrates to implement two different sandwich immunoassay protocols for the detection of mouse monoclonal target antibodies. The magnetic chains form when the chip is placed in a magnetic field, and are geometrically trapped and accurately positioned in a microchannel with periodically enlarged cross-sections. In the first immunoassay protocol, capture and target antibodies are incubated off-chip, while exposure to the detection antibody is performed on-chip. In the second protocol, the complete immunoassay is fully executed on-chip. In the ‘off-chip incubation–on-chip detection’ protocol, antibodies can be detected down to a concentration of 50 ng/mL in a total assay time of 120 min, while consuming 1.5 mL of target antibody solution. Using the full on-chip protocol, our system is able to detect target antibodies in the range of a few ng/mL in 30 min, using only a few tens of nanoliters of target antibody solution and reagents. The ‘off-chip incubation–on-chip detection’ protocol is also applied for dosing antibodies obtained from the supernatant of a cell culture medium.

Springer-Verlag2009

Hydrogel Microfluidics to Control Stem Cell Fate

Steffen Cosson

Biomolecular signaling is of utmost importance in governing many biological processes such as morphogenesis during tissue development where biomolecules regulate key cell-fate decisions. In vivo, these factors are presented in a spatiotemporally tightly controlled fashion and in the context of a soft and hydrated microenvironment. Although state-of-the-art microfluidic technologies allow precise biomolecule delivery in time and space, long-term (stem) cell culture at the micro- scale is far from ideal due to issues related to medium evaporation, limited space for cell growth, shear stress and a lack of cell-instructive microenvironments that might adversely impact cell-fate. As a result, microfluidic cell culture systems are not yet suitable to unravel complex multicellular phenomena. This may explain why they are not yet widely used in biology laboratories. Consequently, the overall goal of this thesis was to overcome this technology gap by developing next generation microfluidics through a combination of microfabrication and smart biomaterials. A special emphasis was placed on decoupling biomolecule presentation at micro-scale from macro-scale cell culture in order to enable long-term stem cell culture within user-friendly multiwell plate formats. First, a novel microfluidic concept was invented to rapidly immobilize linear protein gradients on the surface of poly(ethylene glycol) (PEG)-based hydrogels whose biophysical properties are reminiscent of natural extracellular matrices. This method allows efficient capture of steady-state gradients of tagged proteins (e.g. using Fc or biotin tags) in just a few minutes on engineered hydrogels that display the corresponding auxiliary proteins (e.g. ProteinA or NeutrAvidin). The selectivity and orthogonality of the chosen binding schemes enables the formation of parallel and orthogonal overlapping gradients of multiple proteins, which is impossible using existing platforms. After patterning, the microfluidic chip can be readily removed from the gel for cell culture applications. Using quantitative single-cell time-lapse microscopy, this platform was validated here by probing the effect of fibronectin concentration on the directionality and speed of cell migration. Next, the resolution, flexibility and throughput of the protein patterning on hydrogels was substantially enhanced by the introduction of hydrodynamic flow focusing, that is, the generation of user-defined patterns by spatially controlled step- wise deposition of biomolecules. To this end, a microfluidic device was conceived that enabled the generation of arrays of parallel and crossed overlapping gradients. Application of the platform to generate gradients of immobilized leukemia inhibitory factor (LIF) showed an influence of the LIF concentration on ESC self-renewal. This platform should be useful to systematically probe the effect of biomolecule dose, singly or in combinations, on stem cell behavior in vitro; however, it lacks the ability to dynamically vary the presentation of biomolecules that might be crucial to control stem cell fate towards establishing functional in vitro tissue models. To address this limitation, in the last part of the thesis work a hydrogel-based microfluidic chip was developed that decouples macro-scale cell culture on the gel surface from the precise spatiotemporal biomolecule delivery at the micro-scale, i.e. through the gel layer. The hydrogel chip, used here as a simple insert that is compatible with conventional multiwell plate formats, was optimized to support long- term ESC maintenance in both adherent format and as uniformly sized ESC aggregates termed embryoid bodies. The platform was successfully used for the spatially controlled neuronal commitment of ESC via delivery of gradients of the morphogen retinoic acid. Taken together, in this thesis several innovative microengineered cell culture platforms are presented which enable, perhaps for the first time, the long-term culture and manipulation of stem cell fate at the micro-scale. These systems should be useful to systematically probe in vitro the effect of biomolecule dose and delivery dynamics on stem cell behavior, ultimately facilitating the development of complex in vitro tissue models.